The present invention is directed to methods for screening for fragments and derivatives of Hsp90 that are altered in their subcellular localization, and thus, altered in their activity as compared to wild type Hsp90. The present invention is also directed to screening for modulators of Hsp90 activity as determined by assaying for altered subcellular localization of Hsp90. The present invention is further directed to diagnostic, prognostic and screening uses of Hsp90/centrosome co-purification.
The Hsp90 family of proteins is a group of highly conserved stress proteins that are expressed in all eukaryotic cells, and are essential in yeast and Drosophila. For a general review of heat shock proteins, see Parsell and Lindquist, 1993, Ann. Rev. Genet. 27:437-496. Hsp90 is one of the most abundant proteins in the eukaryotic cell, constituting up to about 1-2% of the cellular protein under normal physiologic conditions, and its expression is increased several-fold in response to stress. Up to now, members of the Hsp90 family were found in the cytosol, the endoplasmic reticulum, and chloroplasts; however, evidence for the actual subcellular localization in the cell or possible association with cellular organelles has so far been cryptic and at best contradictory. Scheibel and Buchner, 1997, The Hsp90 Familyxe2x80x94An Overview, In: Guidebook to Molecular Chaperones and Protein Catalysts, Oxford University Press, pp. 147-151. Although family members of Hsp90 have interchangeable functions, the respective genes are differentially regulated in eukaryotes. Borkovich et al., 1989, Mol. Cell Biol. 9:3919-3930. In most eukaryotic cells, one of the two Hsp90 family members is expressed constitutively at a high level at physiological temperature and is induced only 2-3 times by heat shock. A second family member is expressed at a low basal level at normal temperatures, but its expression is enhanced strongly under restrictive growth conditions, like heat treatment. See, Borkovich et al., 1989, Mol. Cell Biol. 9:3919-3930; Krone and Sass, 1994, Biochem. Biophys. Res. Commun. 204:746-752.
The two genes that encode Hsp90 in humans, Hsp90xcex1 and Hsp90xcex2, are 86% homologous. Further, there is extensive homology with lower species. The 63 kDa Hsp90 homolog in Escherichia coli is 42% identical in amino acid sequence to human Hsp90. The 83 kDa Hsp90 protein homolog of Drosophila (Hsp83) is 78% identical/similar to human Hsp90. See, e.g., Alique et al., 1994, EMBO J. 13:6099-6106; Rebbe et al., 1987, Gene 53:235-245; Blackman et al., 1986, J. Mol. Biol. 188:499-515.
The Hsp90 family has been implicated as an important component of intracellular signaling pathways as well as in assisting protein folding. Dimeric Hsp90 proteins bind molecules such as steroid hormone receptors and the receptor kinases v-src, Raf, and casein kinase II (Catelli et al., 1985, EMBO J. 4:3131-3135; Miyata and Yahara, 1992, J. Biol. Chem. 267:7042-7047; Stancato et al., 1993, J. Biol. Chem. 268:21711-21716; Xu and Lindquist, 1993, Proc. Natl. Acad. Sci, USA 90:7074-7078; Wartmann and Davis, 1994, J. Biol. Chem. 269:6695-6701; van der Straten et al., 1997, EMBO J. 16:1961-1969). In the case of steroid receptors, this interaction is required for efficient ligand binding and transcriptional regulation (Bohen and Yamamoto, 1994, Modulation of Steroid Receptor Signal Transduction by Heat Shock Proteins, In: The Biology of Heat Shock Proteins and Molecular Chaperones, Cold Spring Harbor Laboratory Press, pp. 313-334).
Although the molecular structure of Hsp90 is known in detail and a long list of interacting partners is known, very little is known about the subcellular localization of Hsp90 except that Hsp90 is observed in the cytoplasm and nucleus.
Citation or identification of any reference in Section 2 or in any other section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
The present inventors have discovered that at least a significant portion of the Hsp90 molecules in a cell are localized to, and are a core component of (tightly associated with) the centrosome. Further, the localization of Hsp90 to the centrosome, and thus, its function and role in mitosis and fidelity of chromosome segregation, is conserved over evolution. Hsp90 is known to play important roles in the control of cell signaling and the cell cycle, as well as in transcription, therefore the detection of fragments and derivatives of Hsp90 that have altered localization identifies those Hsp90 molecules that have different activities, and allows for the screening of molecules that alter Hsp90 activity by altering its location.
The present invention is directed to methods of identifying a molecule that alters the centrosomal location of Hsp90 in a cell comprising the following steps in the order stated: (a) contacting the cell with one or more candidate molecules; and (b) detecting localization of Hsp90 molecules in the cell, wherein an increase or decrease in the amount of Hsp90 localized to the centrosome relative to said amount in a cell not so contacted with the one or more candidate molecules indicates that the candidate molecules alter the centrosomal localization of Hsp90. In another embodiment, the present invention is directed to methods of identifying a molecule that alters the centrosomal location of Hsp90 in a cell comprising the following steps in the order stated: (a) recombinantly expressing within the cell one or more candidate molecules; and (b) detecting localization of Hsp90 molecules in the cell, wherein an increase or decrease in the amount of Hsp90 localized to the centrosome relative to said amount in a cell in which the one or more candidate molecules were not so expressed indicates that the candidate molecules alter the centrosomal localization of Hsp90. In a specific aspect of either embodiment, step (b) comprises contacting the cell with an antibody to Hsp90 or a binding region of said antibody, and a fluorescently labeled binding partner of said antibody under conditions conducive to immunospecific binding. In an alternative aspect, the method of detecting comprises contacting the cell with a fluorescently labeled antibody to Hsp90 or a binding region of said antibody under conditions conducive to immunospecific binding. In yet another embodiment, the method of detecting comprises sequencing by mass spectroscopy centrosomal proteins isolated from the cell.
In another embodiment of the present invention, methods of identifying a molecule that affects Hsp90 function in a cell are provided which comprise the following steps in the order stated: (a) contacting the cell with one or more candidate molecules; and (b) detecting or measuring altered centrosomal or chromosomal structure or function, wherein an alteration of centrosomal or chromosomal structure or function relative to said structure or function in a cell not so contacted with the one or more candidate molecules indicates that the candidate molecules affects Hsp90 function. In an alternative embodiment, the method of identifying a molecule that affects Hsp90 function in a cell comprises the following steps in the order stated: (a) recombinantly expressing within the cell one or more candidate molecules; and (b) detecting or measuring altered centrosomal or chromosomal structure or function, wherein an alteration of centrosomal or chromosomal structure or function relative to said structure or function in a cell in which the one or more candidate molecules were not so expressed indicates that the candidate molecules affects Hsp90 function. In a specific aspect of either embodiment, the altered centrosomal or chromosomal structure or function is evidenced by an aberrant mitotic figure, which mitotic figure includes but is not limited to monopolar spindles, aneuploydies, chromosomal missegregation, or chromosome non-joinder.
In yet another embodiment, the present invention is directed to methods of identifying a derivative or fragment of Hsp90 that has altered biological activity relative to wild type Hsp90 comprising detecting a qualitative or quantitative change in intracellular location of said derivative or fragment relative to the intracellular location of wild type Hsp90.
In another embodiment, the method of identifying a molecule that alters the centrosomal location of Hsp90 in a cell comprises the following steps in the order stated: (a) microinjecting into the cell one or more candidate molecules; and (b) detecting localization of Hsp90 molecules in the cell, wherein an increase or decrease in the amount of Hsp90 localized to the centrosome relative to said amount in a cell not so microinjected with the one or more candidate molecules indicates that the candidate molecules alter the centrosomal localization of Hsp90.
The present invention also provides methods for identifying a derivative or fragment of Hsp90 which co-purifies or localizes to the centrosome in a cell comprising the following steps in the order stated: (a) contacting a derivative or fragment of Hsp90 with a cell; and (b) detecting whether said derivative or fragment co-purifies or localizes to the centrosome in said cell. The present invention also provides methods for identifying a derivative or fragment of Hsp90 which co-purifies or localizes to the centrosome in a cell comprising the following steps in the order stated: (a) recombinantly expressing a derivative or fragment of Hsp90 in a cell; and (b) detecting whether said derivative or fragment co-purifies or localizes to the centrosome in said cell. In a specific aspect of these embodiments, step (b) comprises contacting the cell with an antibody to Hsp90 or binding region of the antibody, and a fluorescently labeled binding partner of said antibody under conditions conducive to immunospecific binding. In another specific aspect, step (b) comprises contacting the cell with a fluorescently labeled antibody to Hsp90 or binding region of the antibody under conditions conducive to immunospecific binding. In yet another specific aspect, step (b) comprises sequencing by mass spectroscopy centrosomal peptides isolated from the cell.
In another embodiment of the present invention, methods for screening for a molecule that modulates directly or indirectly Hsp90 activity are provided which comprise the following steps in the order stated: (a) contacting a cell expressing Hsp90 with one or more candidate molecules; and (b) detecting the levels of Hsp90 localized to the centrosome in said cell relative to said levels in a cell not contacted with said candidate molecules, wherein a higher or lower level of Hsp90 localization to the centrosome in the presence of said candidate molecules indicates that the molecules modulate the activity of Hsp90, or which comprise the following steps in the order stated: (a) recombinantly expressing one or more candidate molecules within a cell expressing Hsp90; and (b) detecting the levels of Hsp90 localized to the centrosome in said cell relative to said levels in a cell in which candidate molecules were not so expressed, wherein a higher or lower level of Hsp90 localization to the centrosome in the presence of said candidate molecules indicates that the molecules modulate the activity of Hsp90. In one aspect of this embodiment, Hsp90 localization to the centrosome is detected by a method comprising contacting the cell with a molecule that binds to Hsp90 and a molecule that binds to a centrosome-specific protein other than Hsp90 under conditions conducive to binding, and detecting any binding of the molecules to the same subcellular location that occurs.
In yet another embodiment of the present invention, methods are provided for modulating the activity of Hsp90 by contacting a cell expressing Hsp90 with a molecule that modulates the localization of Hsp90 to the centrosome in a cell.
In yet another embodiment of the present invention, methods are provided for diagnosing or screening for the presence of or a predisposition for developing a disease or disorder characterized by aberrant Hsp90 subcellular localization in a subject by measuring the level of Hsp90 localization to the centrosome in a sample derived from the subject, in which a decrease or increase in the level of centrosomal localization of Hsp90 relative to the level of localization in an analogous sample not having the disease or disorder or a predisposition for developing the disease or disorder indicates the presence of the disease or disorder or the predisposition for developing the disease or disorder.